Promising SARS-CoV-2 main protease inhibitor ligand-binding modes evaluated using LB-PaCS-MD/FMO

Abstract

Parallel cascade selection molecular dynamics-based ligand binding-path sampling (LB-PaCS-MD) was combined with fragment molecular orbital (FMO) calculations to reveal the ligand path from an aqueous solution to the SARS-CoV-2 main protease (M^pro) active site and to customise a ligand-binding pocket suitable for delivering a potent inhibitor. Rubraxanthone exhibited mixed-inhibition antiviral activity against SARS-CoV-2 M^pro, relatively low cytotoxicity, and high cellular inhibition. However, the atomic inhibition mechanism remains ambiguous. LB-PaCS-MD/FMO is a hybrid ligand-binding evaluation method elucidating how rubraxanthone interacts with SARS-CoV-2 M^pro. In the first step, LB-PaCS-MD, which is regarded as a flexible docking, efficiently samples a set of ligand-binding pathways. After that, a reasonable docking pose of LB-PaCS-MD is evaluated by the FMO calculation to elucidate a set of protein-ligand interactions, enabling one to know the binding affinity of a specified ligand with respect to a target protein. A possible conformation was proposed for rubraxanthone binding to the SARS-CoV-2 M^pro active site, and allosteric inhibition was elucidated by combining blind docking with k-means clustering. The interaction profile, key binding residues, and considerable interaction were elucidated for rubraxanthone binding to both M^pro sites. Integrated LB-PaCS-MD/FMO provided a more reasonable complex structure for ligand binding at the SARS-CoV-2 M^pro active site, which is vital for discovering and designing antiviral drugs.

Figure 1

Study overview.

Figure 2

(A) Cell-based and SARS-CoV-2 M^pro inhibition screening; (B,C) effect of rubraxanthone on cell viability, as determined using MTS assay; and (D) Lineweaver–Burk plot of rubraxanthone-induced SARS-CoV-2 M^pro inhibition.

Figure 3

Possible rubraxanthone binding sites in SARS-CoV-2 M^pro, as predicted using BDK. Docked poses and 2D ligand–protein interactions of rubraxanthone (A) and inhibitor AT7519 (PDB code: 7AGA) at free M^pro allosteric site and (B) at substrate-binding M^pro allosteric site.

Figure 4

(A) RMSD of rubraxanthone binding at M^pro and M^pro/substrate complex allosteric sites and corresponding RMSD clustering on last 400 ns trajectories. (B) MM/GBSA per-residue decomposition free energy ($\mathrm{\Delta }{\text{G}}_{\text{bind}}^{\text{residue}}$) for rubraxanthone binding in top two M^pro and M^pro/substrate complex clusters (A-1 and -2 and S-1 and -2, respectively). Residues responsible for ligand binding with $\mathrm{\Delta }{\text{G}}_{\text{bind}}^{\text{residue}}$ ≤ − 1 kcal/mol and/or hydrogen bonding are depicted in right figure panel.

Figure 5

(A) Distance between the ligand and H41–C145 catalytic dyad centres of mass (d_com) plotted for 50 LB-PaCS-MD cycles. (B) Pairwise structural alignment is consistent with the ligand-binding pocket at M^pro active site. (C) Corresponding volume depth compared to that of the SARS-CoV-2 M^pro/X77 complex (6W63) crystal structure.

Figure 6

(A) FMO–RIMP2/PCM interaction energy profile of rubraxanthone binding at M^pro active site. Energy decomposition analysis (PIEDA) and total interaction energy (PIE^Total) are presented as stacked bar graph and grid map, respectively. Key residues of top five complexes exhibiting PIE < − 1 kcal/mol are labelled. (B) Ligand binding distribution in active site, as determined from 100 ns MD simulation, and corresponding d_com is plotted over time.

Figure 7

(A) RMSD, R_g, hydrogen bonding, and d_com of rubraxanthone active-site binding plotted as functions of simulation time. Ligand distribution and top three clusters (P3, P5, and P7), as determined from FMO-based RMSD clustering, are shown above and below plots, respectively. (B) MM/GBSA-calculated binding free energy ($\mathrm{\Delta }{\text{G}}_{\text{bind}}^{\text{MM/GBSA}}$) and energy components for rubraxanthone binding in P3, P5, and P7 clusters are represented by black circles and bar graphs, respectively.

Figure 8

Interaction profile and key binding residues for rubraxanthone binding at M^pro active site in top three clusters: P3-1, P7-1, and P7-3. Residues exhibiting $\mathrm{\Delta }{\text{G}}_{\text{bind}}^{\text{residues}}$ < − 1 kcal/mol are labelled and drawn in right figure panel.